JP2013126381A - Cell fusion method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel cell fusion method in which the cell fusion by one pair of two-cell can be performed more efficiently and more certainly in an electrical cell fusion.SOLUTION: The mixture suspension of cells is introduced in a fusion container that has a micropore that is smaller than the diameter of a cell with a large diameter in the cells that are fused, impressed with an ac voltage, and the introduced cells are induced to the micropores, the cells with a large diameter are fixed first in the micropores, and/or the cells with a small diameter are fixed by the state to touch with the cells, and are impressed with a direct current pulse voltage replacing the ac voltage, and the fusion is performed in 1.0×105-7.0×105 (V/m) of the range of a field intensity that is applied to the touched cells with a small diameter and the cells with large diameter to solve the problem.

Description

  The present invention relates to a method for efficiently fusing two cells having different sizes.

  Conventionally, there is a cell fusion technique in which two different types of cells, such as mouse spleen cells producing a specific antibody and mouse myeloma cells, are fused to form one hybrid cell. It's being used. As a method for fusing cells, an electric cell fusion method is known. In this method, cells are introduced between parallel electrodes to which an AC voltage is applied, the cells are arranged in a bead shape, and a DC pulse voltage of several microseconds to several tens of microseconds is applied between the electrodes in this state. However, there is an advantage that it is possible to fuse cells easily and efficiently without special learning. The electric cell fusion method is suitable for automation, and an apparatus for carrying out the electric cell fusion method has been reported so far.

  The present applicant also has a pair of electrodes made of a conductive member disposed so as to face the cell fusion region of the cell fusion chamber, and a fine electrode disposed between the pair of electrodes and penetrating in the direction of the pair of electrodes. A cell fusion chamber comprising an insulator having a hole has been reported (Patent Document 1, etc.). Explaining the device reported by the present applicant, as shown in FIG. 1, a pair of electrodes made of conductive members arranged so as to face each other in the cell fusion region, and a finely penetrated in the direction of the pair of electrodes. A cell fusion container comprising an insulator having a hole, wherein the insulator is disposed on an electrode surface on the cell fusion region side of one of the electrodes; and the pair of An AC power source for applying an AC voltage to the electrodes and a DC pulse power source for applying a DC pulse voltage. The waveform of the AC voltage applied between the electrodes by the AC power source charges and discharges the cells. Using a cell fusion device having a periodically repeating waveform, the first cell is introduced into the cell fusion region, and the first cell is introduced into the micropore by applying an AC voltage having the waveform. After fixing The second cell is introduced into the cell fusion region, and an AC voltage having the waveform is applied to bring the second cell into contact with the first cell at the position of the micropore. This is a cell fusion method in which cell fusion is performed by applying.

  The cell fusion container is a cell fusion container in which the diameter of a micropore is not more than the diameter of a cell having a large diameter and not less than the diameter of a cell having a small diameter, and the alternating current is used to fix one cell per micropore. In the cell fusion device, a waveform of an alternating voltage applied between the electrodes by a power source is a waveform in which charging and discharging of the cells are periodically repeated by the power source.

Here, the operation of the cell fusion method will be described with reference to FIG. First, as the first cell (22), a cell having a small diameter is introduced into the cell fusion region (1) together with the cell suspension (30), and an alternating voltage having the waveform described above is applied to form one micropore ( Fix one first cell per 9). In this case, the first cell is guided to the micropore mainly by dielectrophoretic force, gravity, and electrostatic force from the electrode. The diameter and depth of the micropore is approximately equal to the diameter of the first cell, and the first cell just enters the micropore. By doing so, electrostatic force is generated on the electrode surface of the bottom surface of the micropore and the first cell, and the first cell is reliably fixed to the micropore. Here, as shown in FIG. 2, the dielectrophoretic force (10) means that the upper electrode (14) and the lower electrode covered with the fine holes (9) when an alternating voltage having a specific frequency is applied between the electrodes. Thus, when there is a concentrated part of the electric force lines (12), it is a force that moves dielectric particles such as cells toward the direction of the concentrated part (11) of the electric lines of force (that is, the direction of the micropores). . In general, the dielectrophoretic force is proportional to the volume of the dielectric particles, the difference between the dielectric constant of the dielectric particles and the dielectric constant of the solution, and the square of the applied voltage, and has a high frequency (for example, 30 kHz or more) for cells and the like. When a voltage is applied, a dielectrophoretic force is generated toward a region where electric lines of force are concentrated.
Next, as the second cell (18), a cell having a large diameter is introduced into the cell fusion region (1) together with the cell suspension (30). At this time, since the first cell (22) is fixed by the electrostatic force with the electrode and is surrounded by the micropore (9), the first cell (22) is fed by feeding the second cell. Cells rarely detach from the micropores. The introduced second cells are contacted and fixed from above the first cells fixed in the micropores by applying the AC voltage having the waveform described above. In this case, the dielectrophoretic force in the micropores also acts on the second cells, but is induced by the first cells fixed in the micropores mainly by gravity and electrostatic force from the first cells. Next, by switching the power source to the DC pulse power source (6) and applying a DC pulse voltage for cell fusion, the first cell and the second cell in contact with each other in the micropore are fused in pairs. Thus, a fused cell (32) can be obtained, and efficient cell fusion in a pair of two cells becomes possible.

  However, the method described in Patent Document 1 enables efficient cell fusion of a pair of two cells, and a high fusion probability is obtained as compared with a normal electric cell fusion method. Among them, there was a problem that the ratio of antibody-producing fused cells that produced the target antibody was not as high as the ratio of improved fusion probability. Here, the fusion probability means (number of fused cells) / (number of spleen cells of BCP immunized mouse used). Also, in the above method, cells that are sized to fit into the micropores are mainly fused, and cells that are larger or smaller than the micropores are difficult to fuse. It was difficult to fuse more efficiently. Further, in the above method, since one of the cells to be fused is introduced into the cell fusion region and fixed in the micropore, the other of the cells to be fused is introduced into the cell fusion region and fixed in the micropore, so that the operation takes time. There is a problem that the cell activity gradually decreases with time.

JP 2007-295912 A

  The object of the present invention has been proposed in view of the conventional situation, and an antibody-producing fused cell that efficiently and reliably fuses two cells by electric cell fusion to produce the target antibody is efficiently produced. It is an object to provide a new cell fusion method for obtaining more target.

As a result of intensive investigations to achieve the above object, the present inventors have completed the present invention. That is, the present invention is a method for fusing two cells having different diameters,
1) Introducing the mixed suspension of cells into a fusion container having micropores smaller than the diameter of the cells to be fused,
2) Inducing introduced cells by applying AC voltage,
3) Fixing the cells in the micropores and fixing the cells in contact with the cells,
4) While applying an alternating voltage or replacing the alternating voltage, apply a DC pulse voltage to fuse the small diameter cell and the large diameter cell,
It is characterized by that.
The present invention will be described below.

  The fusion method of the present invention fuses two cells. The two cells to be fused may be any cells as long as they have different sizes. For example, for the animal species and organs from which they are derived, cells of the same or different origin can be used without limitation ( Hereinafter, of the two cells to be fused, a cell having a diameter larger than the other may be referred to as cell A, and a cell having a smaller diameter than the other may be referred to as cell B).

  In the present invention, the diameter of a cell refers to the diameter when the cell is spherical or nearly spherical, and refers to the diameter of the sphere that is inscribed by the cell when the cell is elliptical or otherwise. As a matter of course, even in the same type of cells, the diameters are slightly different from cell to cell, so the average cell diameter in the cells A or B can be used as the diameter. The cells and sizes used in the present invention are 2-15 μm spleen cells and 5-30 μm cancer cells.

  In addition, as an example of the fusion object in this invention, it can apply also to the liposome and cell, virus, cell, etc. which contain the egg and the sperm, the gene, for example. By fusing them, for example, cells each containing a gene that expresses a different useful protein can be fused to create a single cell that can simultaneously express these two proteins, or for example, to produce a useful antibody And the like, and the cells having proliferative ability are fused to produce proliferative cells while producing useful antibodies.

  Prior to introduction into the fusion container, cells A and B are mixed and made into a suspension in order to introduce two types of cells into the fusion container at the same time, but the suspension components such as mannitol and sucrose As long as it contains components that do not inactivate cells A and B to be fused, such as sugar. The conductivity of the suspension is set to a range of 1 mS or less because dielectrophoretic force needs to act on the cells. In preparing a mixed suspension of cell A and cell B to be introduced into the fusion container, it is particularly preferable to mix cell A and cell B two-to-one. Further, when the fusion container has a plurality of micropores, it is preferable to use at least the number of cells B equal to the number of micropores. This is to maximize the number of cells B fixed in the micropores and obtain a larger number of fused cells by one operation.

In the fusion method of the present invention, when two types of cells having different diameters are introduced into the cell fusion region and the cells are brought into contact with each other at the position of the micropore to perform fusion, the contact surface between the two types of two cells having different diameters It is assumed that a direct-current pulse voltage is applied so that the electric field intensity applied to is preferably 1.0 × 10 ^{5 to} 7.0 × 10 ⁵ (V / m). In this case, more cells that produce the target antibody can be fused, and more antibody-producing fused cells can be efficiently obtained. In comparison, an antibody-producing fused cell acquisition rate of 2 times or more can be obtained. More preferably, a DC pulse voltage is applied so that the electric field strength applied to the contact surface of two types of two cells having different diameters is 2.0 × 10 ^{5 to} 5.0 × 10 ⁵ (V / m). I decided to. In this case, an antibody-producing fused cell acquisition rate of 5 times or more of the antibody-producing fused cell acquisition rate in a normal electric cell fusion method can be obtained. If the electric field strength applied to the contact surface of the two cells is too low, the cells producing the target antibody will not be fused, while if the electric field strength applied to the contact surface of the two cells is too high, the target antibody will be In any case, a desired fused cell cannot be obtained so much, for example, the produced cell itself will die.
In addition, the cell fusion method of the present invention makes it possible to introduce cells A and B to be fused into a cell container in a mixed state so that two types of cells can be simultaneously introduced into the fusion container. . This fusion container has micropores, but the opening of the micropores is smaller than the diameter of the cell A. Here, the opening of the fine hole refers to a portion in contact with the insulator surface of the fine hole. Specifically, for example, when the opening of the micropore is circular, the diameter may be smaller than that of the cell A. Further, specifically, for example, when the opening of the micropore is other than a circular shape, the shape and size of the opening are determined by creating a model or the like in consideration of the flexibility of the cell A to be fused. good. In addition, the fusion container may have a flow path for introducing a suspension in which cells are mixed, and a flow path for discharging the liquid after the method of the present invention is performed outside the container. Instead of forming such a flow path, for example, it is configured to movably attach the surface that becomes the upper lid of the container, and is configured to flip up the upper lid when introducing or discharging the suspension. This can also be illustrated.

  The depth of the micropores is preferably 1/2 or more of the diameter of the cell A. Here, the depth refers to the distance from the opening to the bottom surface of the fine hole. When the depth of the micropore is ½ of the diameter of the cell A, it is the depth at which the cell A enters the bottom of the micropore. By adjusting the depth of the micropores in this way, the cells A are exposed more than half from the micropores, and the cells A and B in the region where the electric field strength is more constant away from the micropore surface. This is because it becomes possible to efficiently perform the fusion with.

When the fusion container has a plurality of micropores, it is particularly preferable that the micropores are arranged at equal intervals with the adjacent micropores. Here, the equal interval means that the distance from the center of an arbitrary microhole to the center of an adjacent microhole is equal. This is because, as will be described later, when an alternating voltage is applied to the cells in the fusion container and induced into the micropores, the electric lines of force are concentrated uniformly in each micropore, causing cell fusion in all the micropores. Because. As a specific example of arranging the adjacent fine holes at equal intervals, for example, it can be illustrated like a grid pattern (hereinafter, such an arrangement may be described as an array or array arrangement). it can).
In the present invention, the distance between adjacent micropores is particularly preferably 0.5 to 6 times the diameter of the cell A, and particularly preferably about 1 to 2 times the diameter of the cell A. This is because the electric field generated when the AC voltage is applied can be made uniform by arranging the fine holes at equal intervals. In addition, if the interval between the micropores is too narrow, the probability that a plurality of cells are fixed in one micropore is increased. On the other hand, if the interval between the micropores is too wide, cells are left between the micropores. This is because there is an increase in wasted cells that do not contribute to fusion or become desired fusion cells in any case, such as the possibility increases.

The fusion container having the fine holes described above can be configured in various forms and shapes. As will be described later, in the present invention, an operation is performed in which an alternating voltage is applied to the cells in the fusion container to induce the micropores. For this reason, in this invention, although an electrode is arrange | positioned in the vicinity of a fusion container, when making an electrode and a fusion container contact, a fusion container is comprised with insulating materials, such as glass, a ceramic, resin. Examples of the shape include a cylindrical shape, a cone shape, and a column shape. In general, considering that the volume of the mixed suspension introduced into the fusion container for fusion is about several hundred μL to 1 ml, for example, a box-like one with a bottom of 2 cm × 2 cm and a height of about 1 mm Is preferred. In order to efficiently induce the cells in the mixed suspension by applying an alternating voltage described later, when the mixed suspension is introduced into the fusion vessel, the mixed suspension in the cell induction direction is introduced. It is preferable that the height (width) of the liquid is about 0.5 mm to 2 mm. Also, if the height (width) of the mixed suspension in the cell induction direction is too low, the cells will sink to the bottom before filling the cells into the fusion container, and the cells will be uniformly distributed in the fusion container. On the other hand, if the height (width) of the mixed suspension in the cell induction direction is too high, it takes a long time for the cells to settle gradually due to gravity and be induced to micropores by dielectrophoretic force. This requires efficiency.
By the way, the cell fusion container may be perpendicular or parallel to the gravity. However, since the cell moves not only by the dielectrophoretic force but also by the electrostatic force from the gravity or the electrode, the influence of the movement other than the dielectrophoresis due to the application of the alternating voltage is applied. It is particularly preferable to provide the micropores in the direction of gravity (usually the bottom wall of the fusion vessel) for the purpose of reducing the amount.

  The fine hole of the fusion container may be, for example, a through hole or a closed hole provided in the side wall or bottom wall of the fusion container. May be configured using another member. As an example of configuring the micropores using a member different from the fusion container, specifically, for example, a plate-like member may be provided with a through hole and disposed on the bottom surface of the fusion container. In any case, when an alternating voltage is applied by an electrode to be described later, in order to guide the cells in the mixed cell suspension to the micropores, the electric lines of force pass through the micropores and into the fusion container. In addition, the arrangement of the fine holes, the material of the member forming the fine holes, the arrangement of the electrodes, and the like are taken into consideration.

In the cell fusion method of the present invention, an alternating voltage is first applied to the mixed suspension of cells introduced into the fusion container. For this reason, in the present invention, an electrode for applying a voltage is used, but the electrode to be used is not particularly limited. For example, the structure which arrange | positions two flat plates which each arrange | positioned an electrode so that a fusion container may be pinched | interposed can be illustrated. For example, the structure which arrange | positions one flat plate which has arrange | positioned a pair of electrode on the same plane in the lower part of a fusion container can also be illustrated. (For example, FIG. 16)
In the present invention, first, an alternating voltage is applied to the cells A and B in the cell mixture suspension to induce and fix the cells in the micropores. In order to apply an AC voltage, the above-described electrode may be connected to a known AC power source. The waveform of the applied AC voltage is determined by charging and discharging cells in the fusion container by applying the AC voltage. Is a waveform having one or more times within a half period in which a voltage having a value other than 0 preferably does not change for a certain period of time. Preferably, an alternating voltage whose waveform is a rectangular wave, a trapezoidal wave, or a combination thereof is applied. By applying such an alternating voltage, the cells A and B in the mixed suspension are induced into the micropores. In the present invention, when an AC voltage is applied, the AC voltage preferably has no DC component. This is due to the electrostatic force generated by the DC component causing the cells to move in response to a biased force in a specific direction, which prevents the cells from being fixed in the micropores due to the dielectrophoretic force or is contained in the mixed suspension. This is because the ionic component generates an electric reaction on the electrode surface and heat is generated, which may cause the cell to perform thermal movement and control the movement of the cell due to the dielectrophoretic force. When the fusion container has a plurality of micropores, the micropores are arranged in an array, and electrodes having dimensions and shapes that cover all of the arranged micropores are used. It is preferable that an electric field is generated almost uniformly in all the fine holes by the applied voltage.

  By adding an alternating voltage having the waveform described above, preferably an alternating voltage having a waveform in which charging and discharging of the cells in the fusion vessel are periodically repeated, the cells in the mixed suspension are made into micropores by dielectrophoresis. Can be attracted. The applied voltage value and frequency may be set appropriately depending on the distance between the electrodes, the type and size of cells to be fused, and the type of mixed suspension.

  By applying an AC voltage, the cells A and B are fixed in contact with the micropores. Therefore, both cells can be fused by applying a DC pulse voltage while applying an AC voltage or in place of the AC voltage. When applying a DC pulse voltage while applying an AC voltage, an electrode for applying a DC pulse voltage is used separately from the electrode for applying an AC voltage. The electrode can also be used as a DC pulse voltage application electrode.

When a DC pulse voltage is applied, the electrical conductivity of the cell membrane of the contacting cell instantaneously decreases, and the cell membrane composed of the lipid bilayer is reversibly disrupted and reconstituted to fuse both. . In the fusion method of the present invention, as described later, it is highly likely that the cell A is first fixed in the micropore, and then the cell B is fixed on the cell A in contact with the cell A. Here, in general, the smaller the cell diameter, the higher the DC pulse voltage that causes reversible disturbance of the cell membrane. Therefore, as described above, the cell A is equal to or more than the number of micropores and the cell B is equal to the number of micropores. The mixed suspension is preferably used and the DC pulse voltage is adjusted appropriately to eliminate the fusion of cells A or B, induction of three or more cells, etc. It becomes possible to efficiently obtain a fused cell in which the cell B is fused on a one-to-one basis. In addition, when a suspension in which cells A are mixed at least three times the number of micropores and cells B is mixed with the number of micropores is used, cells B may not easily enter the arrayed micropores. More preferably, a suspension in which the cell A is twice the number of micropores and the cell B is mixed with the same number of micropores is used. In this case, there is a high possibility that two cells A are continuously fixed on the micropore and then the cell B is fixed. When a DC pulse voltage is applied, the cell A fixed on the surface of the micropore is Since the cell membrane is destroyed and ruptured by the strong electric field strength, the contact surface between the cell A fixed on the cell membrane and the cell membrane of the cell B in contact with the cell A is further away from the surface of the micropore. The electric field strength at the point approaches a constant value compared to the surface of the micropore, and only the fusion of the two can be realized.

According to the present invention, the following effects can be obtained.
(1) In the cell fusion device of the present invention, the insulator in which the micropores are formed is arranged on one of the electrode surfaces on the cell fusion region side, so that the cells are securely fixed in the micropores, and the vicinity of the micropores The two cell pairs in the cell can be selectively fused with each other, and the cell fusion with the two cell pairs can be performed efficiently.
(2) In the cell fusion method of the present invention, two types of cells having different diameters are mixed and introduced into the cell fusion region, and an alternating voltage is applied to fix and contact the cells in the micropores. The cell fusion is performed by applying a direct-current pulse voltage. By doing so, the operation can be performed easily and in a short time, and further, the cell fusion can be performed while maintaining the cell activity.
(3) In the cell fusion method of the present invention, two types of cells having different diameters are introduced into the cell fusion region, the cells are brought into contact at the position of the micropore, and the two types of cells having different diameters are contacted. By fusing the electric field strength applied to the surface in the range of 1.0 × 10 ^{5 to} 7.0 × 10 ⁵ (V / m), more cells that produce the target antibody can be fused. It becomes possible to efficiently obtain more production fusion cells, and an antibody production fusion cell acquisition rate of 2 times or more can be obtained as compared with the antibody production fusion cell acquisition rate in the usual electric cell fusion method. Further, by applying a DC pulse voltage such that the electric field strength applied to the contact surface of two types of two cells having different diameters is 2.0 × 10 ^{5 to} 5.0 × 10 ⁵ (V / m). An antibody-producing fused cell acquisition rate that is 5 times or more of the antibody-producing fused cell acquisition rate in a normal electric cell fusion method can be obtained.
(4) In the cell fusion method of the present invention, when two types of cells having different diameters are fused, the concentration of the cells is more than twice the number of micropores, and the smaller diameter cells are finer. It is more preferable that the number of holes be equal, and in this way, even if there is a range of cell sizes, the DC pulse voltage can be adjusted appropriately so that the number of holes can be increased under a certain electric field strength. Thus, it becomes possible to selectively and efficiently fuse many cells in pairs of two cells.
(5) In the cell fusion device of the present invention, the diameter of the planar shape of the micropore is smaller than the diameter of the cell having the larger diameter among the two types of cells to be fused. It can be securely fixed by the hole.
(6) In the cell fusion method of the present invention, the depth of the micropore is ½ or more of the diameter of the cell A, and the cells A and B are mixed and introduced into such a cell fusion region, This is a method of immobilizing the cells A in the micropores by applying an AC voltage, bringing the cells B into contact with the micropores, and applying a DC pulse voltage to fuse the cells. Thus, since the contact surface of the cell membrane of cell A and cell B is separated from the surface of the micropore, fusion occurs under a constant high electric field strength, and more cells can be efficiently fused in a pair of two cells. Is possible.
(7) In the cell fusion device of the present invention, a plurality of micropores are formed in an array on an insulator. By doing so, a pair of two cells fixed in the plurality of micropores can be simultaneously obtained. Cell fusion is possible, and cell fusion in a pair of two cells can be performed efficiently.
(8) In the cell fusion device of the present invention, the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cells to be inserted into the micropore. The probability of fixing one cell in the micropore can be increased, and the probability of bringing two cells into contact with each other in the micropore can be increased.
(9) In the cell fusion device of the present invention, it is possible to fix one cell in one minute hole by controlling the waveform of the AC voltage applied between the electrodes by the AC power source.

It is a conceptual diagram explaining sectional drawing and operation | movement of the chamber for cell fusion of patent document 1. FIG. It is a figure for demonstrating the dielectrophoresis used for this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a conceptual diagram of an example of the cell fusion apparatus suitable for implementing this invention, and the cell fusion apparatus used in the Example. FIG. 4 is an AA ′ cross-sectional view of the cell fusion container shown in FIG. 3. It is the figure which showed the sine wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the triangular wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the trapezoid wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the rectangular wave as an example of the waveform of the alternating voltage used for this invention. It is the 1st figure for explaining the present invention. It is a 2nd figure for demonstrating this invention. It is a 3rd figure for demonstrating this invention. It is a figure which shows the example of the cell fusion method of this invention. It is the figure which showed the electric field strength of a micropore vicinity. It is the schematic of general photolithography and the etching method. It is a figure showing the relationship between the electric field intensity concerning the contact surface of two cells in the cell fusion method of this invention, and an antibody production fusion cell acquisition rate. As an example of the cell fusion device used in the present invention, it is a conceptual diagram of a cell fusion device in which a single flat plate having a pair of electrodes arranged on the same plane is arranged at the bottom of a fusion container.

1: Fusion region 2: Electrode 3: Conductive wire 4: Power supply 5: AC power supply 6: DC pulse power supply 7: Changeover switch 8: Insulator 9: Fine hole 10: Dielectrophoretic force 11: Concentration part of electric force line 12: Line of electric force 13: Fusion container 14: Upper electrode 15: Lower electrode 16: Spacer 17: Microhole A '
18: Cell A
19: Inlet 20: Outlet 21: Fine hole B '
22: Cell B
23: ITO
24: Glass 25: Resist 26: Photomask for exposure 27: Exposure 28: Insulator-integrated lower electrode with fine holes 29: Developer 30: Cell suspension 31: Peak voltage 32: Fusion cell 33: Electrode a
34: Electrode b
35: Comb electrode

  In the following, an embodiment of the present invention will be described with reference to the drawings in order to explain the present invention in more detail.

  FIG. 3 shows a conceptual diagram of an apparatus that can be used to carry out the cell fusion method of the present invention. This apparatus is roughly divided into a fusion container (13) and a power source (4). The fusion vessel is constructed integrally with the electrode, and a spacer (16) is arranged between the upper electrode (14) and the lower electrode (15), and the insulator (8) in which the fine holes (9) are formed is provided at the lower part. It has the structure arrange | positioned at the cell fusion region side of the electrode. The material of the upper electrode and the lower electrode is not particularly limited as long as it is a conductive member and is a chemically stable member. An alloy such as platinum, gold, copper or an alloy such as stainless steel or ITO (Indium Tin Oxide) Although a glass substrate or the like on which a transparent conductive material such as tin) is formed may be used, in order to observe the cell fusion state, it is preferable to use a glass substrate on which a transparent conductive material such as ITO is formed as an electrode. .

  There are no particular limitations on the dimensions of the upper electrode and the lower electrode, but a size that is easy to handle is preferably, for example, a size of about 70 mm long × 40 mm wide × 1 mm thick.

  The spacer is used to prevent the upper electrode and the lower electrode from coming into direct contact and to secure a space for holding the cell mixture suspension in the fusion container. Insulators are arranged. The material should just be an insulating material, for example, glass, ceramic, resin etc. can be illustrated. In addition, the spacer includes an introduction channel for introducing cells into the fusion container and discharging the suspension after the fusion and an introduction port (19) communicating with the introduction channel, a discharge channel and a discharge communicating with the introduction channel. An outlet (20) can also be provided.

  The size of the spacer is not particularly limited as long as the upper electrode and the lower electrode are not in contact with each other, but a size that can effectively use the entire surface of the electrode is preferable. For example, if the electrode size is approximately 70 mm long × 40 mm wide, the size of the spacer is, for example, approximately 40 mm long × 40 mm wide. The region portion (1) that essentially functions as a fusion container is a space portion inside the spacer formed by the insulator formed with the spacer, the upper electrode, and the fine holes, and the spacer that creates this space portion. For example, when the size of the spacer is about 40 mm long × about 40 mm wide, the space inside the spacer is about 20 mm long × 20 mm wide. The thickness of the spacer may be about 0.5 to 2.0 mm.

  A fine hole (9) is formed in the insulator (8) disposed under the spacer. The material of the insulator (8) is not particularly limited as long as it is an insulating material such as glass, ceramic, resin, etc., but since it is necessary to form a through hole, it is relatively easy to process the resin or the like. Are preferred. As a means for forming fine holes penetrating the resin, a method of irradiating a laser at the position of the fine hole to be formed, or a method of molding using a mold having a pin for forming the through hole at the position of the fine hole A known method such as the above may be used. In addition, when UV curable resin or the like is used for the insulator, a fine hole penetrating through general photolithography (exposure) and etching (development) using an exposure photomask on which a pattern corresponding to the fine hole is drawn is formed. Can be formed. When forming a plurality of fine holes in the insulator, it is preferable to form the fine holes by a general photolithography and etching method using a UV curable resin for the insulator.

  FIG. 4 is a schematic view showing an AA ′ cross-sectional view of the fusion container of FIG. 3. As a means of bonding the upper electrode (14), the spacer (16), the insulator (8), and the lower electrode (15) as shown in FIG. 4, each of them is bonded with an adhesive and heated in a pressurized state. Known methods are used, such as a method of fusing, and a method in which the spacers are bonded together by being made using a resin such as PDMS (poly-dimethylsiloxane) having a surface adhesive property or a silicon sheet. That's fine By doing in this way, the area | region (1) which is a substantial fusion container shown in FIG. 4 can be formed.

  A power source (4) is connected to the upper and lower electrodes of the cell container via a conductive wire (3). The power source (4) applies an AC voltage waveform between the upper electrode (14) and the lower electrode (15), and a DC pulse voltage for cell fusion between the upper electrode and the lower electrode. The AC power source (5) and the DC pulse power source (6) can be used by appropriately switching them using a switching mechanism such as a selector switch (7).

  The AC power source used in the cell fusion method of the present invention is not particularly limited as long as the cells can be fixed in the micropores. For example, a sine wave, a rectangular wave, a triangular wave, a trapezoidal wave, etc. with a peak voltage of about 1 V to 20 V and a frequency of about 30 kHz to 3 MHz. An AC power source that can output the AC voltage is acceptable.

In the cell fusion method of the present invention, it is possible to perform more efficient cell sorting by fixing one cell in one micropore, but AC for fixing one cell per one micropore. The voltage waveform is preferably a rectangular wave. The reason for this is that, as shown in FIGS. 5 to 8, the waveform of the AC voltage is instantaneous compared to the sine wave (FIG. 5), the triangular wave (FIG. 6), and the trapezoidal wave (FIG. 7). Since the cell quickly moves into the micropores because the peak voltage (31) set in is reached, the probability that the cells overlap and enter the micropores is low, and thus the probability of fixing one cell per micropore Becomes higher. In addition, cells can be regarded as capacitors electrically, and while the peak voltage of the rectangular wave does not change, it is difficult for current to flow into the cells that have entered the micropores. Since the dielectrophoretic force is less likely to be generated in the micropores, once a cell enters the micropore, the probability that another cell enters the micropore is reduced, and electric lines of force are generated to generate dielectrophoretic force. The cells sequentially enter the empty micropores, and the cells can be uniformly fixed in all the micropores arranged in an array in the cell fusion region.
In addition, in the waveform of an alternating voltage in which the voltage continuously changes like a sine wave or a triangular wave, there are micropores where a plurality of cells are concentrated and fixed, and micropores where the cells are not fixed at all. It is difficult to fix one cell per hole and to fix cells uniformly in all micropores.
Moreover, it is preferable that the waveform of the alternating voltage used with the cell fusion method of this invention does not have a direct-current component. This is because the cells move by receiving a biased force in a specific direction due to the electrostatic force generated by the DC component, and it becomes difficult to fix the cells in the micropores due to the dielectrophoretic force. Ions contained in the suspension cause an electrical reaction on the electrode surface, generating heat, causing the cells to undergo thermal motion, and the cell movement cannot be controlled by the dielectrophoretic force, attracting the cells to the micropores. This is because it becomes difficult.
The DC pulse power source used in the cell fusion method of the present invention is particularly limited as long as it can generate a DC pulse voltage capable of cell fusion of the first cell and the second cell that are in contact with each other at or near the micropore. For example, there is no particular limitation as long as it is a DC pulse power supply capable of outputting a DC pulse voltage of about 50 V to 1000 V and a pulse width of about 10 μsec to 50 μsec as the DC pulse voltage.
In the cell fusion method of the present invention, the diameter of the planar shape of the micropore penetrating the insulator is smaller than the diameter of the cell fixed to the micropore, and the interval between the plurality of micropores is equal to the diameter of the cell to be fixed. In the range of 0.5 to 6 times, in a plurality of micropores formed in an array on an insulator, one cell is fixed per micropore and cells are uniformly distributed in all micropores It can be fixed.
FIG. 9 to FIG. 11 show conceptual diagrams of processes in which cells enter micropores in the cell fusion method of the present invention. The thickness of the insulator (8) is larger than ½ of the diameters of the cells A (18) and B (22), and the inner diameters of the micropores are smaller than the diameters of the cells X and Y. Further, it is assumed that after the cell X enters the micropore A ′ (17) as shown in FIG. 10, the cell Y enters the micropore B ′ (21) as shown in FIG.

  The cell fusion method of the present invention uses the cell fusion apparatus having the above-described characteristics to apply and mix the cells A and B by applying the AC voltage as described above, so that all the micropores are introduced. Fix the cells uniformly and contact another cell at the location of the micropores. Here, since the cell A has a larger diameter than the cell B, when mixed and introduced into the fusion container, the cell A tends to sink earlier than the cell B due to gravity, and the dielectrophoretic force is equal to the radius of the cell. Since it is proportional to the third power, the cell A is more likely to be fixed in the micropore before the cell B. For this reason, due to gravity and dielectrophoretic force, the cells A are first fixed in the micropores, and the cells B are easily fixed thereon. On the other hand, when the cell A is fixed to one micropore and then the cell B is fixed from above the fixed cell A, the cell B is subjected to dielectrophoretic force, gravity, and the electrostatic force of the cell A. Contact with cell A. However, for the reasons described above, since the cells A block the micropores, the current does not easily flow, so that the generation of electric lines of force is suppressed, and the dielectrophoretic force acting on the cells B is weakened. Therefore, the probability that the cells B are brought into contact with the cells A fixed to the micropores one by one is lowered. On the other hand, when the cells A and B are mixed and introduced and an AC voltage is applied, the concentration (number) of the cells A is made higher (more) than the concentration (number) of the cells B, so By introducing, it becomes possible to improve the contact probability between the cells A and B and the acquisition rate of cells producing the target antibody.

  As shown in FIG. 12, the suspension vessel (30) in which the cell A (18) and the cell B (22) are mixed is introduced into the fusion vessel (1), and an AC voltage having the waveform described above is applied. Then, the cells are fixed in the micropores (9). In this case, the cells are guided to the micropores mainly by dielectrophoretic force, gravity, and electrostatic force from the electrodes, and the cells come into contact with each other and are fixed to the micropores by gravity and electrostatic force from each cell. The cell A having a larger diameter is more likely to be fixed before the cell B having a smaller diameter. Here, the number of cells A is not particularly limited, but considering the effective use of cells A, the number is preferably equal to the number of micropores. Next, the power source is switched to a direct current pulse power source (6), and a direct current pulse voltage for cell fusion is applied to cause cell A and cell B in contact with each other in the micropores to be fused in a pair of cells, so that fused cells (32) can be obtained.

  Since electric field lines are concentrated in the fine holes, the electric field strength in the vicinity of the fine holes has the highest electric field strength on the electrode surface in the fine holes, as shown in FIG. 13, from the insulating film surface to the other electrode. The electric field strength gradually decreases. FIG. 13 shows a finite element method for calculating the electric field strength when an arbitrary voltage is applied between electrodes by arranging one minute hole having an arbitrary diameter and depth in an insulating film of an arbitrary thickness on one electrode. It is calculated using. The vertical axis is the value obtained by normalizing the electric field intensity with the maximum electric field intensity, and the horizontal axis is the position between the electrodes. There is an electrode in which an insulating film is arranged at the origin of the horizontal axis. The insulating film surface corresponds to the position indicated by the dotted line in the figure, and the range from the origin of the horizontal axis to the dotted line corresponds to the insulating film thickness. In the calculation performed this time, the electric field strength on the surface of the micropore decreases as the distance from the surface of the micropore decreases, as shown in FIG. 13, without depending on the material and thickness of the insulating film and the size and depth of the micropore.

  In general, as described above, in the electric cell fusion method, by applying a DC pulse voltage for cell fusion, the electric conductivity of the cell membrane is instantaneously reduced, and the reversible disturbance of the cell membrane composed of lipid bilayers. And the cell is fused by reconfiguration. In general, the smaller the cell diameter, the higher the direct-current pulse voltage causing reversible disturbance of the cell membrane. For this reason, the cell A and the cell B are preferably paired with the cell A having the larger diameter and the cell B having the smaller diameter. The suspension mixed at a ratio of 1 is introduced into the fusion container, the AC voltage having the waveform described above is applied to fix the cells A and B in the micropores in this order, and a DC pulse voltage of an appropriate voltage is applied. Thus, it is possible to efficiently obtain a fused cell in which cell A and cell B are fused one-on-one. Further, as shown in FIG. 13, the electric field strength gradually decreases and becomes constant as the distance from the surface of the micropores becomes constant, so that the electric field strength at the contact surface between the cell membranes of cell A and cell B is constant compared to the surface of the micropores. Get closer to. In this way, when cells A and B having different diameters are fused, even if there is a range of cell sizes, by adjusting the DC pulse voltage appropriately, Thus, it becomes possible to efficiently fuse more cells. Conversely, for this purpose, the cell concentration (number) is preferably 2 to 1 between cell A and cell B, and cell A is twice the number of micropores ( It is preferable that the cell B be equal to the number of micropores). For reference, even if two cells A are continuously fixed on the micropore and the cell B is fixed, the cells fixed on the surface of the micropore when the DC pulse voltage is applied. In A, the cell membrane is destroyed and ruptured by a strong electric field strength, and the contact surface between the cell A immobilized on the cell membrane and the cell membrane of the cell B in contact with it is further away from the surface of the micropores. The electric field strength at the contact surface is closer to that of the surface of the micropore, and only the fusion of the two can be realized.

  Hereinafter, embodiments of the present invention will be described in detail. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the scope of the invention.

  FIG. 3 shows a conceptual diagram of the cell fusion device used in Example 1. The cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 3, the cell fusion container has an insulator (8) in which a spacer (16) is arranged between an upper electrode (14) and a lower electrode (15), and a plurality of micropores are formed in an array. It has a structure sandwiched between a spacer and a lower electrode.

The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Further, as shown in FIG. 3, an introduction port (19) and a discharge port (20) for introducing and discharging a cell suspension containing cells were provided. The insulator (8) having a plurality of fine holes is provided with a fine hole in which the lower electrode (15) and the insulator in which the plurality of fine holes are formed in an array are integrally formed by the photolithography and etching method shown in FIG. An insulator-integrated lower electrode (28) was used.
First, a resist (25) was applied to the ITO film-forming surface of Pyrex (registered trademark) glass (24) on which ITO (23) was formed using a spin coater so as to have a film thickness of 2.5 μm. After drying, pre-baking (80 ° C., 15 minutes) was performed using a hot plate. A xylene negative resist was used as the resist. Next, in the area of 30 mm length × 30 mm width, a micro hole pattern with a diameter of 8.5 μm arranged in an array of 1500 vertical x 1500 horizontal, with the vertical and horizontal spacing of the fine holes being 20 μm is drawn. The resist was exposed (27) with a UV exposure machine using the exposure photomask (26), and developed with a developer (29). The exposure time and development time were adjusted so that the depth of the micropores was 5 μm, which was equal to the film thickness of the resist, so that the ITO was exposed on the bottom surface of the micropores. After development, the resist was hardened by post-baking (115 ° C., 30 minutes) using a hot plate. The upper electrode (14), the spacer (16), and the insulator-integrated lower electrode (28) with fine holes were laminated and pressure-bonded as shown in FIG. FIG. 4 is a cross-sectional view of the cell fusion container shown in FIG. 3 taken along the line AA ′. The surface of the silicon sheet was sticky, and the parts were brought into close contact with each other by pressure bonding, and the cell suspension containing the cells could be put into the cell fusion container without leakage. Since the area where the spacer is hollowed is 20 mm long × 20 mm wide, the number of micropores existing in this space is about 1 million. The power source for applying a voltage between the electrodes is a signal generator (NF circuit design block, WF1966) as an AC power source, and a cell fusion power source (Neppagene, LF101) as a DC pulse power source via a conductive wire (3). The AC power supply and DC pulse power supply can be switched to the electrode by a changeover switch.
Here, the diameter of the micropore of the insulator-integrated lower electrode with micropore (28) is smaller than the diameter of the cell having a larger diameter among the two types of cells to be fused.

BCP-immunized mouse spleen cells (φ5 μm) and mouse myeloma cells (φ10 μm) immunized with BCP (Blue Carrier Immunogenic Protein) antigen were used. BCP antibody-producing cells are present in mouse spleen cells immunized with BCP antigen. BCP immunized mouse spleen cells and mouse myeloma cells are mixed 1: 2 and both cells are suspended in an aqueous mannitol solution at a concentration of 300 mM, and the cell suspension is made to a density of 5.0 × 10 ⁶ cells / mL. The liquid was adjusted. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

  First, 600 μL of the cell suspension (BCP immunized mouse spleen cell number: about 1 million cells, mouse myeloma cell number: about 2 million cells) was injected from the introduction port of the spacer using a syringe, and a voltage of 20 Vpp, When a rectangular wave AC voltage with a frequency of 3 MHz is applied between the electrodes, BCP immunized mouse spleen cells and mouse myeloma cells can be fixed in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. It was possible to arrange a plurality of cells in an array.

Next, the power supply is switched to a DC pulse power supply (LF101, manufactured by Nepagene Co., Ltd.), cell fusion is performed by applying a DC pulse voltage with a voltage of 250 V and a pulse width of 30 μs between the electrodes. The cell suspension in the container was placed in a HAT medium (H: hypoxanthine, A: medium containing aminopterine, T: thymidine), and the fused cells were cultured. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cell culture was performed. After 6 days, the number of fused cells was counted. As a result, 1000 fused cells could be confirmed. A fusion probability of 10/10000 per piece was obtained. Here, the fusion probability represents (number of fused cells) / (number of spleen cells used in BCP immunized mice). Moreover, the supernatant was taken from the HAT medium in which the obtained fused cells were cultured, and the number of fused cells producing BCP antibody among the fused cells was counted by enzyme immunoassay (ELISA). As a result, 12 BCP antibody productions were obtained. Fusion cells could be confirmed, and an antibody-producing fusion cell acquisition rate of 12/1000000 was obtained for 1 million spleen cells of all injected BCP immunized mice. Here, the antibody-producing fused cell acquisition rate represents (number of BCP antibody-producing fused cells) / (number of spleen cells of BCP immunized mice used).
This is because the fusion probability is 50 times the fusion probability 0.2 / 10000 in normal electric cell fusion shown in Comparative Example 1 described later, and the antibody-producing fusion cell acquisition rate 0.19 / in normal electric cell fusion. The antibody-producing fused cell acquisition rate was 63 times that of 1,000,000, and it was confirmed that efficient cell fusion in a pair of two cells and efficient acquisition of antibody-producing cells were confirmed. In addition, antibody production in electrofusion performed by fixing the cell A on the micropore using the two types of cells having different diameters shown in Comparative Example 2 and then fixing the cell B from above the cell A The antibody production fusion cell acquisition rate was 4 times as high as the fusion cell acquisition rate of 3 / 1,000,000, and efficient acquisition of antibody production cells could be confirmed.
In addition, the BCP immunized mouse spleen cells and mouse myeloma cells shown in Comparative Example 3 were mixed at a ratio of 1: 1, introduced into the cell fusion region, and the cells were fixed in the micropores by applying an alternating voltage. The antibody production fusion cell acquisition rate was 6 times higher than the antibody production fusion cell acquisition rate of 2 / 1,000,000 in the method of performing fusion, and it was possible to confirm the efficient acquisition of antibody production cells.
Further, for the fusion probability 4.2 / 10000 and the antibody-producing fused cell acquisition rate 8/1000000 in the electrofusion in the cell fusion device having the cell fusion region sandwiched between the parallel electrodes having no micropores shown in Comparative Example 4 However, this method shows a high value for both fusion probability and antibody-producing fused cell acquisition rate, and this method using a cell fusion device provided with micropores enables cell fusion that can use cells more effectively and efficiently. It has become possible.

Further, in Example 1, when the DC pulse voltage was changed between 100 V and 600 V, the electric field strength applied to the contact surface of two types of two cells having different diameters was plotted on the horizontal axis for all injected BCP immunity. FIG. 15 shows a graph in which the vertical axis represents the antibody-producing fused cell acquisition rate for mouse spleen cells. Thus, when fusion is performed in the range of 2.0 × 10 ^{5 to} 5.0 × 10 ⁵ (V / m), the antibody is 40 times higher than the probability of antibody-producing fused cells in normal electric cell fusion. Produced fusion cell probability was obtained.

(Comparative Example 1)
For comparison, normal electrical cell fusion was performed. A gold wire electrode (MS gold wire electrode, manufactured by Nepagene Co., Ltd.) having a distance between electrodes of 1 mm was used as an electrode for performing electric cell fusion, and a cell fusion power source (manufactured by Nepagene, LF101) was connected to this electrode.

BCP immunized mouse spleen cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Mouse antibody-producing cells and mouse myeloma cells are mixed at a ratio of 4: 1. Both cells are suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension is adjusted to a density of 5 × 10 ⁷ cells / mL. did. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

800 μL of the cell suspension (BCP immunized mouse spleen cell number: about 32 million cells, mouse myeloma cell number: about 8 million cells) was injected between the electrodes, and a cell fusion power source was used with a voltage of 20 Vpp and a frequency of 3 MHz. After confirming the formation of the cell pearl chain by applying a sinusoidal AC voltage between the electrodes, a DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, 640 fused cells were confirmed, and 32 million total mouse antibody-producing cells were produced. A fusion probability of 0.2 / 10000 was obtained. Moreover, the supernatant was taken from the HAT medium in which the obtained fused cells were cultured, and the number of fused cells producing BCP antibody among the fused cells was counted by enzyme immunoassay (ELISA). As a result, 6 BCP antibody productions were obtained. Fusion cells could be confirmed, and an antibody-producing fusion cell acquisition rate of 0.19 / 1000000 was obtained for 32 million spleen cells of all BCP immunized mice.

(Comparative Example 2)
As a comparative example, in the cell fusion device of Example 1, when two types of cells having different diameters are used and the diameter of the cell A is larger than the diameter of the cell B, the cell A is fixed in the micropore, Cell fusion was carried out by fixing the cell B from above A.

BCP immunized mouse spleen cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in 300 mM aqueous mannitol solution, and BCP immunized mouse spleen cells had a density of 1.7 × 10 ⁶ cells / mL, and mouse myeloma cells had 3.4 × 10 ⁶ cells / mL. Each cell suspension was adjusted so as to have a density of. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

  First, 600 μL of the mouse myeloma cell suspension (number of mouse myeloma cells: about 2 million) was injected from the introduction port of the spacer using a syringe, and a rectangular wave AC voltage with a voltage of 20 Vpp and a frequency of 3 MHz was applied by an AC power source. When applied between the electrodes, one mouse myeloma cell can be fixed to each of a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds, and the plurality of cells are arranged in an array. I was able to.

  Subsequently, 600 μL of the BCP immune mouse spleen cell suspension (number of mouse antibody-producing cells: about 1 million cells) was applied to the spacer while a rectangular wave AC voltage of 20 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power source. It was injected using a syringe from the inlet.

Next, the power supply is switched to a DC pulse power supply (LF101, manufactured by Nepagene Co., Ltd.), cell fusion is performed by applying a DC pulse voltage with a voltage of 250 V and a pulse width of 30 μs between the electrodes. The cell suspension in the container was placed in a HAT medium, and the fused cells were cultured. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, 800 fused cells could be confirmed, and all BCP-immunized mouse spleen cells had 1 million cells. A fusion probability of 8/10000 per piece was obtained. In addition, the supernatant was taken from the obtained HAT medium in which the fused cells were cultured, and the number of fused cells producing BCP antibody among the fused cells was counted by enzyme immunoassay (ELISA). Fusion cells could be confirmed, and an antibody-producing fusion cell acquisition rate of 3/1000000 was obtained for 1 million spleen cells of all BCP-immunized mice.

(Comparative Example 3)
As a comparative example, in the cell fusion apparatus of Example 1, BCP immunized mouse spleen cells and mouse myeloma cells were mixed at a ratio of 1: 1, introduced into the cell fusion region, and an AC voltage was applied to the cells in the micropores. Was fixed and cell fusion was performed in the same manner.
BCP immunized mouse spleen cells and mouse myeloma cells were mixed 1: 1, and both cells were suspended in a 300 mM aqueous mannitol solution to a density of 3.4 × 10 ⁶ cells / mL. A cell suspension was prepared.
To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

  First, 600 μL of the above cell suspension (BCP immunized mouse spleen cell number: about 1 million cells, mouse myeloma cell number: about 1 million cells) was injected from the introduction port of the spacer using a syringe, and a voltage of 20 Vpp, When a rectangular wave AC voltage with a frequency of 3 MHz is applied between the electrodes, BCP immunized mouse spleen cells and mouse myeloma cells can be fixed in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. It was possible to arrange a plurality of cells in an array.

Next, the power supply is switched to a DC pulse power supply (LF101, manufactured by Nepagene Co., Ltd.), cell fusion is performed by applying a DC pulse voltage with a voltage of 250 V and a pulse width of 30 μs between the electrodes. The cell suspension in the container was placed in a HAT medium, and the fused cells were cultured. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, 600 fused cells could be confirmed, and all BCP immunized mouse spleen cells had 1 million cells. A fusion probability of 6/10000 per piece was obtained. Moreover, the supernatant was taken from the HAT medium in which the obtained fused cells were cultured, and the number of fused cells producing BCP antibody among the fused cells was counted by enzyme immunoassay (ELISA). Fusion cells could be confirmed, and an antibody-producing fusion cell acquisition rate of 2 / 1,000,000 was obtained for 1 million spleen cells of all BCP-immunized mice.

(Comparative Example 4)
As a comparative example, in the same cell fusion apparatus as in Example 1, BCP immunized mouse spleen cells and mouse myeloma cells were 1 using a cell fusion container having a cell fusion region sandwiched between parallel electrodes having no micropores. : The mixture was mixed at 2, introduced into the cell fusion region, an pearl chain was formed by applying an alternating voltage, and cell fusion was performed in the same manner.
Here, both the upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick.
In addition, BCP immunized mouse spleen cells and mouse myeloma cells were mixed at a ratio of 1: 2, and both cells were suspended in an aqueous mannitol solution having a concentration of 300 mM, resulting in a density of 5.0 × 10 ⁶ cells / mL. The cell suspension was adjusted as follows.
To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

  First, 600 μL of the cell suspension (BCP immunized mouse spleen cell number: about 1 million cells, mouse myeloma cell number: about 2 million cells) was injected from the introduction port of the spacer using a syringe, and a voltage of 20 Vpp, When a rectangular wave AC voltage with a frequency of 3 MHz was applied between the electrodes, BCP immunized mouse spleen cells and mouse myeloma cells formed a pearl chain and lined up in the electrode direction in an extremely short time of about 2 to 3 seconds.

  Here, the pearl chain refers to a phenomenon in which cells are arranged in a bead shape.

Next, the power supply is switched to a DC pulse power supply (LF101, manufactured by Nepagene Co., Ltd.), cell fusion is performed by applying a DC pulse voltage with a voltage of 250 V and a pulse width of 30 μs between the electrodes. The cell suspension in the container was placed in a HAT medium, and the fused cells were cultured. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cell culture was performed. After 6 days, the number of fused cells was counted. As a result, 420 fused cells could be confirmed. A fusion probability of 4.2 / 10000 per piece was obtained. Moreover, the supernatant was taken from the obtained HAT medium in which the fused cells were cultured, and the number of fused cells producing BCP antibody among the fused cells was counted by enzyme immunoassay (ELISA). The fused cells could be confirmed, and an antibody-producing fused cell acquisition rate of 8/1000000 was obtained for 1 million spleen cells of all BCP immunized mice.

Claims (4)

A method of fusing two types of cells with different diameters,
1) Introducing the mixed suspension of the two types of cells into a fusion container having micropores smaller than the diameter of the cells having a larger diameter among the cells to be fused,
2) Introducing cells introduced by applying an AC voltage into the micropores,
3) fixing the cells in the micropores and / or fixing the cells in contact with the cells;
4) Instead of an alternating voltage, a DC pulse voltage is applied to fuse a small diameter cell and a large diameter cell,
A cell fusion method characterized by the above. Two types of cells having different diameters are introduced into the cell fusion region, the cells are brought into contact with each other at the position of the micropore, and the electric field strength applied to the contact surface between the two types of two cells having different diameters is 1.0 × E. The cell fusion method according to claim 1, wherein fusion is performed in the range of ^{5 to} 7.0 × E ⁵ (V / m). The cell fusion method according to claim 1 or 2, wherein the micropore has a depth of ½ or more of a diameter of the cell having a large diameter. 4. The cell according to any one of claims 1 to 3, wherein the cells having a large diameter are at least twice the number of micropores, and the cells having a small diameter are mixed with the number of micropores and introduced into the cell container. The cell fusion method described.

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